Expression of heterologous nucleic acids in various biological systems is a necessary tool in the study of gene regulation as well as a powerful technique in the development of agricultural varieties of plants, algae, fungi, and animals which possess improved traits. DNA or RNA may be introduced, for instance, into cells for transient gene expression wherein the introduced nucleic acids remain episomal and are not integrated into the replicating genetic material of the host cell. Transient expression of heterologous nucleic acids is valuable for testing the functional level of regulatory sequences in directing native RNA polymerases to precisely transcribe the gene of interest and polyadenylate the RNA transcript. Transient gene expression is also valuable to test the level and fidelity of sense or antisense transcription, translation and ultimately the functionality of the heterologous gene product.
Transgenic organisms, on the other hand, comprise plants and animals that have heterologous nucleic acid sequences artificially integrated as functional addendum to their natural genetic repertoire. Transgenic organisms also comprise plants and animals that have antisense heterologous nucleic acids integrated into their genetic repertoire to effect the attenuation of natural or artificial gene expression. See, e.g., U.S. Pat. No. 5,175,385 to Wagner et al.; U.S. Pat. No. 5,185,384 to Krimpenfort et al.; U.S. Pat. No. 5,175,383 to Leder et al.; U.S. Pat. No. 4,736,866 to Leder et al.; and U.S. Pat. No. 5,107,065 Anti-Sense Regulation of Gene Expression in Plant Cells. A successfully produced transgenic organism permanently contains the heterologous nucleic acid sequence stably integrated in a non-deleterious manner into its native genetic composition and is able to pass the corollary trait on to its natural progeny. Techniques are needed for efficient transformation of single cells, cells comprising tissues, and the production of transgenic organisms.
Functional nucleic acid sequences are introduced into living cells in various ways including well known methods using calcium phosphate and DEAE-dextran and polybrene mediated transfection, protoplast fusion, and introduction via cationic liposomes. Cells and tissues from different sources contrast sharply in their ability to take up and express exogenously added DNA and RNA. Irrespective of the method used to introduce nucleic acids into eucaryotic and procaryotic cells, the efficiency of transient or stable transformation and gene expression is determined largely by the cell and tissue type that is used.
The innate ability of the natural plant pathogen Agrobacterium tumefaciens to incorporate its own DNA into certain plant genomes has been widely utilized to transfer foreign genes into plants by artificial engineering of the natural transferred portion of the pathogen plasmid DNA. Agrobacterium-mediated plant cell transformation has been found to be successful in a limited number of dicot plants including tobacco, petunia and carrot. Unfortunately, important agronomic monocotyledonous crops such as wheat, asparagus, barley, rye, corn and canola are generally not susceptible to transformation by Agrobacterium tumefaciens. The production of transgenic plants via Agrobacterium tumefaciens is further limited by the applicability of this method to species where one or a few transformed cells, e.g. leaf cells, can be regenerated into whole plants by means of artificial hormonal stimuli. Methods exist for regeneration of relatively few agronomic dicots and are virtually non-existent for monocots.
Methods used for transformation of plant cells also include electroporation and microinjection. To transform living cells by electropotation, cells are subjected to an electric shock to cause those cells to uptake DNA from surrounding fluid. Although protoplasts from such monocotyledonous plants as corn and rice have been successfully transformed by electroporation, current methods for the regeneration of whole monocot crop plants are limited to certain varieties. The need for genetic transformation of corn and wheat as well as many other agronomically important crops exists due to the potential to improve their disease resistance and output traits.
Methods of microinjection are described in U.S. Pat. No. 5,255,750, Microinjection Apparatus and Method of Controlling Microinjection; U.S. Pat. No. 5,114,854 Process of Effecting a Microinjection Into Living Cells; and U.S. Pat. No. 4,743,548 Plant Cell Microinjection Technique. Microinjection is a tedious process that requires microscopic manipulation of single cells (usually very large cells such as oocytes and plant cells without cell walls) and therefore is not practical for gene implant into plant tissues.
One modification of microinjection involves pricking the cell nuclei with a solid glass needle to allow biological solutions to enter which surround the cell (Yamamoto, M. et al., Exp. Cell Res., 142:79-84 (1982)). The pricking of single cell nuclei has at least the same limitations as microinjection and has only been demonstrated in mouse fibroblast nuclei.
U.S. Pat. No. 2,309,391, A Device for Injectively Treating Plants, describes a method of macro-injection wherein a large hand-held device artificially fertilizes or infects large plants, such as trees, by means of injection of fluids through hollow needles.
More recently, plant tissue transformation has been produced through the use of particle-mediated "gene gun" technology. See, e.g. Sanford, The Biolistic Process TIB-TECH, 6:299-302 (1988); and U.S. Pat. No. 5,204,253 Method and Apparatus for Introducing Biological Substances into Living Cells. According to such "gene gun" technology, DNA or RNA is coated on micro spherical carrier particles of a dense metal, e.g., tungsten, gold or platinum. The carrier particles are accelerated to physically pierce and imbed within a living target tissue to carry nucleic acid into the tissue. A number of different mechanisms have been employed to accelerate and project the coated carrier particles into target tissue including gunpowder ignition, pressured gas and a shock wave created by electric shock.
In practice, it has been difficult to accurately control the delivery and distribution of the coated carrier particles using such particle accelerating mechanisms. This has usually resulted in highly inefficient transformation of the desired microscopic growth regions in target tissues. For example, the distribution pattern of the micro particles often comprise a spaced pattern of dense clusters of coated particles in the target tissue accompanied by multiple particle entries per cell which often have deleterious or lethal physical effects upon the target cells and tissue. In other instances, the distribution pattern of coated particles may be too sparse to produce any useful cell transformations in a particularly valuable target tissue such as a meristem tissue of a plant, even after many attempts at site specific impregnation in the target tissue. Further, "gene gun" methods presently call for use of a vacuum (which can damage tissue by expansion or desiccation), and are implemented with expensive and complex systems and apparatus requiring complex calculations, settings, configurations and experimentation in order to operate the systems with any degree of success using various biological tissues.
Therefore, there is a continuing need for a relatively simple and inexpensive instrument and method for precisely controlled site-specific delivery of biological substances to the cytoplasm of a wide variety of tissue cells in situ. The present invention satisfies that need.